Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here I developed greatly miniaturized microfluidic devices for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells (MSC). The devices were integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional image acquisition. Since primary stem cells can be isolated only in limited number, I optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter cell culture volumes and automated microfluidic controls for pulsed medium supply, my platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by preferential movement of MSC in serum gradients and by using a CXCL/CXCR ligand/receptor combination in a co-culture design. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens.